| The genus Citrus L.is considered to have originated from the southeast piedmonts of the Himalayas,a region that includes the Yunnan province.In the last few hundred years,it has been introduced to tropical and subtropical areas where climate warm and fit for the growth of citrus.Citrus tristeza disease that caused by citrus tristeza virus(CTV)is one of the most serious citrus diseases,which is widely distributed in citrus growing areas all over the world and causes great economic losses many times.CTV is the only known citrus-infecting closterovirid while grapevine,cherry,and blackberry all can be infected by more than one closterovids.Our knowledge of citrus viruses is largely skewed toward virus pathology in cultivated orchards while little is known about the virus diversity in natural ecosystems.Here,we identified five genotypes of CTV and three novel closterovirids from the citrus samples collected in wild habitat using viral metagenomics.In this study,grafting transmission test,sequence verification,genomic structure,characteristics analysis,evolution and recombination analysis were carried out for the virus in wild citrus.Further,we investigated the symptoms on different citrus varieties for one of the new viruses and established its real-time fluorescence quantitative PCR detection system.The main results of this study are as follows:1.This study was based on the analysis of r RNA-depleted RNA libraries from 32 wild citrus tree samples collected in the Ailao Mountain region in Yunnan Province between July 2018 and August 2019.From these samples,we identified five genotypes of CTV and three putative novel closterovirids.Each potential viral genome was further examined by RT-PCR and rapid amplification of c DNA methods.All three novel viruses had the capacity to be graft-transmitted to citrus trees.Field investigation showed that CaAV-1 and CiVB were distributed in Dehong Prefecture and Binchuan County of Yunnan Province.CaAV-1 showed leaf-roll associated symptoms on different citrus varieties.2.Genomic organization and characteristics analysis showed the newly identified citrus-infecting closterovirids have the following characteristics: i)are highly divergent compared to known members of the family,ii)have an extraordinary ability to recombine with different sources and,iii)vary widely in their genome organization and expression strategy.We found that frequent horizontal gene transfer,gene duplication,and alteration of expression strategy have shaped the genome complexity and diversification of the family Closteroviridae.3.Phylogenetic analysis showed that CaAV-1 and CaAV-2 are new members of the genus Ampelovirus,and CiVB may represent a new genus in the family Closteroviridae.Recombination frequently occurred among distinct Closteroviridae members,thereby facilitating the evolution of Closteroviridae.CaAV-1 and CTV have similar asymmetrical variation genome — a conserved 3’-half moiety and a more variable5’-half counterpart.Due to the limits of technology,early research in wild citrus plant pathology only analyzed molecular characteristics of incomplete genomic sequences of CTV.Our research represents the first time of virus diversity investigation in wild citrus.Given the significant negative impact of CTV on the citrus industry worldwide and the potential of new emerging virus infections to affect citrus growers,this work laid a foundation for the study of the biological characteristics and epidemic risk of these new closterovirids and points out the importance of conservation of the wild citrus resource.A few closterovirus-derived genes and RNA interference vectors have been developed to control plant diseases and these novel citrus viruses may be suitable for the construction of new vectors for citrus disease control and character improvement. |
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